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Fig. 1. Schematic drawing of the cop operon and model of copper homeostasis in E. hirae. Copper(I) is taken up by CopA under copper-limiting conditions. Inside the cell, CopZ complexes copper(I) to safely deliver it to the CopY repressor, which regulates expression of the cop operon. If intracellular copper is excessive, CopZ delivers copper to CopB for secretion.

2.1. CPx-Type ATPases

The structure and function of copper ATPases has become one of the focal points of research on copper homeostasis. Copper ATPases differ significantly in their primary structure, membrane topology, and evolutionary relationship from the previously known P-type ATPases, such as the Ca2+-ATPases or the Na+K+-ATPases (Fig. 2). They thus form a distinct subclass that has been called Pj-type ATPases (6) or CPx-type ATPases based on the conserved intramembranous motif CPC or CPH (7). Bacterial cadmium ATPases, silver ATPases, and the Escherichia coli zinc ATPase are close relatives of the copper ATPases and these heavy-metal ATPases are also members of the CPx-type ATPase subclass (10-12). CPx-type ATPases are highly conserved from bacteria to man and must have arisen very early in evolution, probably before the division of prokaryotes and eukaryotes (13). Figure 3 shows a phylogenetic tree of representative members of the CPx-type heavy metal and the P-type non-heavy-metal ATPase families.

2.1.1. The E. hirae CopA ATPase

CopA of E. hirae exhibits 43% sequence identity with the human Menkes and Wilson ATPases; in the transduction domain, sequence identity between these enzymes is even 92% (15). This suggests that CopA is a representative model of a copper ATPase. Based on indirect evidence, CopA appears to function in copper uptake. Cells disrupted in cop A cease to grow in media in which the copper has been complexed with 8-hydroxyquinoline or o-phenanthroline. This growth inhibition could be overcome by adding copper to the growth media. Interestingly, null mutants in copA could grow in the presence of 5 pM AgNO3, conditions that fully inhibit the growth of wild-type cells. Thus, the CopA ATPase provides a route for the entry of copper, but also silver into the cell (1). Silver transport by CopA is probably fortuitous, as silver has no known biological role. The transport of Ag(I) by CopA would indicate that Cu(I) rather than Cu(II) is transported by CopA.

CopA could be expressed in E. coli and purified to homogeneity by Ni-NTA affinity chromatography by means of an added histidine tag. Figure 4 shows the single-step purification of CopA from E. coli extracts on a Ni-NTA agarose column, eluted with an imidazole gradient. Purified CopA was active and had a pH optimum of 6.3 and a Km for ATP of 0.2 mM. The enzyme formed an acylphosphate intermediate, which is a hallmark of P- and CPx-type ATPases (16). This purified

Fig. 2. Comparison of the membrane topology of a CPx-type ATPase and a P-type ATPase. Shown are CopB (A) of E. hirae and the Ca2+-ATPase of sarcoplasmic reticulum (B). Helices common to both type of ATPases are in black and helices unique to one type of ATPase are in gray. Key sequence motifs are indicated in the one-letter amino acid code and the numbers denote the position of residues in the sequence. In the center of the figure, the approximate locations of the three cytoplasmic domains A, P, and N are indicated. MBD, metal-binding domain containing repeat metal-binding sites; TGE, conserved site in transduction domain A; CPx, putative copper-binding site; DKTGT, phosphorylation site in domain P; HP, motif of unknown function, probably in domain N; GDG, nucleotide-binding site residues in domain N.

Fig. 2. Comparison of the membrane topology of a CPx-type ATPase and a P-type ATPase. Shown are CopB (A) of E. hirae and the Ca2+-ATPase of sarcoplasmic reticulum (B). Helices common to both type of ATPases are in black and helices unique to one type of ATPase are in gray. Key sequence motifs are indicated in the one-letter amino acid code and the numbers denote the position of residues in the sequence. In the center of the figure, the approximate locations of the three cytoplasmic domains A, P, and N are indicated. MBD, metal-binding domain containing repeat metal-binding sites; TGE, conserved site in transduction domain A; CPx, putative copper-binding site; DKTGT, phosphorylation site in domain P; HP, motif of unknown function, probably in domain N; GDG, nucleotide-binding site residues in domain N.

Fig. 3. Phylogram of the CPX-type ATPases with a selected sample of P-type ATPases. Divergence was scored by the Jukes-Cantor method (50). Relationships between distant branches are not reliable.

Fig. 4. Purification of CopA. Extract from E. coli expressing CopA endowed with a 6xhis tag was bound to a Ni-NTA agarose column and eluted with an imidazole gradient. Fractions were analyzed on a 10% polyacry-lamide gel, stained with Coomassie blue. The arrowhead shows the position of purified CopA.

Fig. 4. Purification of CopA. Extract from E. coli expressing CopA endowed with a 6xhis tag was bound to a Ni-NTA agarose column and eluted with an imidazole gradient. Fractions were analyzed on a 10% polyacry-lamide gel, stained with Coomassie blue. The arrowhead shows the position of purified CopA.

preparation of CopA can now serve to analyze mechanistic aspects of copper transport and to characterize structure-function relationships.

2.1.2. The E. hirae CopB ATPase

CopB differs from CopA and Menkes/Wilson-type copper ATPases mainly at the N-terminus. Instead of a CxxC metal-binding motif, CopB features a histidine-rich N-terminus similar to the one observed in the zinc ATPase of E. coli (9). Wild-type cells of E. hirae can grow in the presence of up to 6 mM CuSO4. The CopB ATPase was found to be required for this copper-resistant growth. Null mutation of copB rendered the cells sensitive to copper, whereas null mutation of cop A had no significant effect on the copper tolerance. This suggested that the CopB ATPase is a copper-export ATPase, extruding excess copper from the cytoplasm and thus conferring copper resistance. Using 64Cu+ and 110mAg+, CopB was shown to catalyze ATP-driven accumulation of copper(I) and silver(I) in native membrane vesicles. Uptake of copper by these vesicles would correspond to copper extrusion in whole cells. Use of null mutants in either copA, copB, or cop A and copB allowed one to clearly assign the observed transport to the activity of the CopB ATPase. Copper transport exhibited an apparent Km for Cu+ of 1 |M and a Vmax of 0.07 nmol/min/mg of membrane protein. 110mAg+ was transported with similar affinity and rate (17). However, because Cu+ and Ag+ were not free in solution but complexed to Tris-buffer and dithiothreitol under the experimental conditions, the Km values must be considered as relative only. The results with membrane vesicles were further supported by 110mAg+ extrusion from whole cells loaded with this isotope. Again, transport depended on the presence of functional CopB (18). Vanadate showed an interesting biphasic inhibition of ATP-driven copper and silver transport: Maximal inhibition of Cu+ transport was observed at 40 |M VO43- and of

Ag+ transport at 60 |M VO43-. Higher concentrations relieved the inhibition of transport. This behavior is unexplained at present, but may relate to the complex chemistry of vanadate involving many oxidation states (19).

For the purification of CopB for further functional analysis and crystallization attempts, a strain for the overproduction of the protein was engineered. Y1 is a repressor-deficient strain that overexpresses CopA and CopB about 50-fold (20). CopB could be extracted from Y1 membranes with dodecyl-P-D-maltoside and purified by Ni-NTA chromatography by means of the endogenous metal-binding capacity of CopB. This single-step purification removed the majority of all contaminating proteins. Final purification was achieved by anion-exchange chromatography on Mono Q Sepharose (21,22).

When CopB was reconstituted into Asolectin proteoliposomes by the method of Apell et al. (23), a threefold increase in ATPase activity was observed. Fifty to eighty percent of this activity could be inhibited by vanadate. Reconstituted CopB was shown to form an acylphosphate intermediate, and thus to exhibit active turnover (22). Using this model system, it was possible to analyze the significance of individual amino acid residues for their functional involvement in catalysis. The Menkes disease mutation C1000R, which changes the conserved CPC motif in membrane helix 6, was mimicked in CopB with the C396S mutation. This mutant CopB ATPase was unable to restore copper resistance in a CopB knockout strain in vivo. The purified C396S ATPase still formed an enzymephosphate intermediate, but had no detectable ATPase activity. The Wilson's disease mutation H1069Q, which is the single most frequent mutation in Europe, was introduced into CopB as H480Q. This mutant CopB similarly failed to restore copper resistance in a CopB knockout strain. Purified H480Q CopB formed an acylphosphate intermediate and retained significant ATPase activity (24). These findings show that S396 and H480 of CopB are key residues for ATPase function, which suggests similar roles for S1000 and H1069 in the Menkes and Wilson ATPase, respectively. The results also suggested that these mutations do not directly affect the site of ATP binding and phos-phorylation.

3. THE CopY REPRESSOR

The two copper ATPases of E. hirae are induced by ambient copper. Induction of the genes is lowest in standard growth media (copper content = 10 |M). If the media copper is increased, an up to 50-fold induction is observed at 2 mM extracellular copper. Full induction is also obtained by 5 |M Ag+ or 5 |M Cd2+. The induction by silver and cadmium is, in all likelihood, fortuitous, because it does not confer resistance to these highly toxic metal ions. Because CopA serves in copper uptake and CopB in its extrusion, this coinduction of CopA and CopB by high and low copper seems puzzling at first. However, it may be a safety precaution: If the cells would express, under copper-limiting conditions, only the import ATPase, they would become highly vulnerable to copper poisoning in case of a sudden increase in ambient copper.

The regulatory gene, copY, upstream of the genes encoding the CopA and CopB ATPases, was cloned by chromosome walking. CopY encodes a repressor protein of 145 amino acids (20). As shown in Fig. 5A, the N-terminal half of CopY exhibits around 30% sequence identity to the bacterial repressors of P-lactamases, Mecl, PenI, and Blal (25-27). In the best studied of these, PenI, this N-terminal portion appears to be the domain that recognizes the operator (28). At position 31, there is a diglutamine motif in CopY. This motif is also found in the phage 434 and lambda Cro repressors at a similar position. By X-ray crystallography, it could be shown that the diglutamine motif of these phage repressors interact with the ACA motif in the DNA with an extraordinarily tight fit. Because the CopY DNA-binding sites feature ACA motifs, it appeared likely that they interact with the QQ motif in CopY (Fig. 5B). Although there is no significant sequence homology between the phage repressors and CopY, they appeared to be a good model.

Because both, the 434 and the Cro repressor are dimeric, the aggregation state of CopY in solution was investigated. It could be shown by crosslinking as well as by size-exclusion chromatography that

Fig. 5. Structural features of the CopY repressor. (A) The N-terminal part of CopY exhibits sequence homology to the P-lactamase repressors of MecI, PenI, and BlaI, whereas the C-terminal portion features cysteine residues that are probably involved in copper binding. (B) Schematic drawing of the putative interaction of the QQ motif of CopY with the ACA triplet in the promoter.

Fig. 5. Structural features of the CopY repressor. (A) The N-terminal part of CopY exhibits sequence homology to the P-lactamase repressors of MecI, PenI, and BlaI, whereas the C-terminal portion features cysteine residues that are probably involved in copper binding. (B) Schematic drawing of the putative interaction of the QQ motif of CopY with the ACA triplet in the promoter.

Fig. 6. Seizing of CopY by gel permeation chromatography. Rf values of the indicated standard proteins and of purified CopY were determined on a TSK-100 column.

CopY is a dimer in solution (Fig. 6). By DNaseI fingerprinting and by band-shift assays, it was shown that CopY interacts at two discrete sites on the promoter, featuring an inverted repeat (29). Presumably, each one CopY dimer bound to each of the sites. The two CopY binding sites also featured two ACA triplets each, suggesting that each CopY monomer interacts with an ACA sequence. A possible interaction of CopY with ACA was investigated by site-directed mutagenesis of the promoter. It could be shown that the affinity of CopY for binding sites mutated from ACA to

TCA was strongly reduced. When both CopY binding sites of the inverted repeat were mutated ACA to TCA, the operator became hyperinducible by low copper concentrations (30).

In the C-terminal half of CopY, there are multiple cysteine residues, arranged as CXCX4CXC. The consensus motif CXCX4-5CXC is also found in the three yeast-copper-responsive transcriptional activators, ACE1, AMT1, and MAC1 (31-33) and appears to be the copper-binding site of the repressor.

Disruption of the E. hirae copY gene results in constitutive overexpression of the cop operon in vivo (20). Binding of CopY to an inverted repeat sequence upstream of the copY gene has been demonstrated in vitro. Thus, CopY appears to be a copper-responsive repressor protein with an N-terminal DNA-binding domain and a C-terminal copper-binding domain.

CopY was overexpressed in E. coli and purified to near homogeneity. The interaction of the purified repressor with the promoter region was shown in band-shift assays as follows: DNA fragments of 530 base pairs encompassing the putative promoter region were incubated with purified repressor protein. The formation of DNA-protein complexes was visualized by the change in electrophoretic mobility of the radioactively labeled DNA band on polyacrylamide gels. Increasing concentrations of CopY lead to a shift of the DNA band. This shift occurred in two steps, suggesting that two monomers or two multimers of the repressor interact with the promoter sequence. Competition experiments with either cold promoter DNA or DNA carrying the promoter of the Na+H+-antiporter gene of E. hirae clearly demonstrate that CopY binding to the cop promoter is specific (29).

Thus, the combined evidence of CopY binding to promoter DNA in vitro and the observed hyperinducibility of promoter mutations in the ACA triplets in vivo points to the following mechanism of regulation: If intracellular copper is in the physiological range, CopY is bound to the promoter and transcription of the cop operon is turned off. If cytoplasmic copper is increased, CopY is released from the promoter and the expression of the cop genes is turned on. But how does CopY sense the cytoplasmic copper level? The answer to this question came from the study of the CopZ chaperone, discussed next.

4. THE CopZ COPPER CHAPERONE

It has recently been discovered that the intracellular delivery of copper to copper-utilizing enzymes requires the action of specialized proteins, the so-called chaperones (34). In E. hirae, the 69-amino-acid protein CopZ fulfills this role. CopZ-like copper chaperones have also been described in humans (HAH1), C. elegans (CUC-1), and yeast (ATX1) (35-37). The conserved domains feature a universal CxxC metal-binding motif and exhibit sequence similarity over a region of 50-60 amino acids (Fig. 7). Interestingly, the N-termini of heavy-metal-binding proteins, such as copper ATPases, cadmium ATPases, and mercuric reductases also contain one to six copies of the conserved copper chaperone sequence (7,38,39). Figure 8 schematically shows the occurrence of CopZ-like building blocks in a number of enzymes involved in heavy-metal metabolism. Clearly, there has been the evolution of a heavy-metal-binding motif that can function either as an isolated unit as in CopZ or as a component of a larger heavy-metal-binding protein. These CopZ-like building blocks in the copper ATPases have been shown to bind copper ions (40-42). However, whether these copper-binding sites function as an integral part of enzyme catalysis or whether they fulfill a more accessory role in scavenging metal ions or regulating enzyme activity remains to be shown.

4.1. Intracellular Copper Routing

CopZ is so far the only chaperone for which copper transfer has been shown directly in vitro. Purified Zn(II)CopY binds to a synthetic cop promoter fragment in vitro (Fig. 9). CopZ was shown to specifically deliver copper(I) to Zn(II)CopY, thereby releasing CopY from the DNA. It could also been shown by luminescence spectroscopy that two copper(I) were thereby quantitatively transferred from Cu(I)CopZ to Zn(II)CopY, with displacement of the zinc(II) and transfer of copper from a nonluminescent exposed binding site in CopZ to a luminescent solvent-shielded binding site in

CopZ ----------- ~MKQEFSVKG MSCNHCVARI EEAVGRI. SG VKKVKVQLKK EKAWKFDEA

HAH1 ----------MPKHEFSVD. MTCGGCAEAV SRVLNKL. .G GVKYDIDLPN KKVCIE... S

ATX1 -------MAE IKHYQFNW. MTCSGCSGAV NKVLTKLEPD VSKIDISLEK QLVDVY...T

. CUC-1 ----------~MTQYVFEMG MTCNGCANAA RKVLGKLGED KIKIDDINVE TKKITVTTDL

Menkes ----MDPSMG VNSVTISVEG MTCNSCVWTI EQQIGKV.NG VHHIKVSLEE KNATIIYDPK

MerP ---VAPVWAA TQTVTLAVPG MTCAACPITV KKALSKV. EG VSKVDVGFEK REAWTFDDT

CCC2 ---------- MREVILAVHG MTCSACTNTI NTQLRAL.KG VTKCDISLVT NECQVTYDNE

Cop A -------MATN TKMETFVITG MTCANCSARI EKELNEQ.PG VMSATVNLAT EKASVKYTDT

CadA MSEQKVKLME EEMNVYRVQG FTCANCAGKF EKNVKKI. PG VQDAKVNFGA SKIDVYGNAS Mer A ----------—MTHLKITG MTCDSCAAHV KEALEKV. PG VQSALVSYPK GTAQLAIVPG

Fig. 7. Alignment of the conserved domain of CopZ with related metal binding motifs. EMBL/GenBank accession numbers are given in parentheses. CopZ, copper chaperone of E. hirae (Z46807); HAH1, human copper chaperone (U70660); ATX1, yeast copper chaperone (L35270); CUC-1, C. elegans copper chaperone (AB017201); Menkes, copper-binding motif of human Menkes ATPase (L06133); MerP, periplasmic mercury-binding protein (P04129); CCC2, copper-binding motif of yeast CCC2 copper ATPase (L36317); CopA, copper-binding motif of E. hirae CopA copper ATPase (L13292); CadA, cadmium-binding motif of Staphylococcus aureus cadmium ATPase (J04551); MerA, mercury-binding motif of mercuric reductase (A00406). The asterisks denote the universally conserved cysteine residues.

Fig. 8. Schematic representation of the occurrence of CopZ-like motifs in various proteins. The polypeptide chains are drawn to scale as boxes. Transmembranous helices are indicated by open rectangles and CopZ-like building blocks by filled rectangles.
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